Three-dimensional electron microscopy reconstruction and cysteine-mediated crosslinking provide a model of the type III secretion system needle tip complex.

Type III secretion systems are found in many Gram-negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the device's external injection needle. The Shigella tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild-type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three-dimensional images of TCs at ∼ 20 Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease-protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies.

Updates on the web-based VIOLIN vaccine database and analysis system.

The integrative Vaccine Investigation and Online Information Network (VIOLIN) vaccine research database and analysis system (http://www.violinet.org) curates, stores, analyses and integrates various vaccine-associated research data. Since its first publication in NAR in 2008, significant updates have been made. Starting from 211 vaccines annotated at the end of 2007, VIOLIN now includes over 3240 vaccines for 192 infectious diseases and eight noninfectious diseases (e.g. cancers and allergies). Under the umbrella of VIOLIN, >10 relatively independent programs are developed. For example, Protegen stores over 800 protective antigens experimentally proven valid for vaccine development. VirmugenDB annotated over 200 'virmugens', a term coined by us to represent those virulence factor genes that can be mutated to generate successful live attenuated vaccines. Specific patterns were identified from the genes collected in Protegen and VirmugenDB. VIOLIN also includes Vaxign, the first web-based vaccine candidate prediction program based on reverse vaccinology. VIOLIN collects and analyzes different vaccine components including vaccine adjuvants (Vaxjo) and DNA vaccine plasmids (DNAVaxDB). VIOLIN includes licensed human vaccines (Huvax) and veterinary vaccines (Vevax). The Vaccine Ontology is applied to standardize and integrate various data in VIOLIN. VIOLIN also hosts the Ontology of Vaccine Adverse Events (OVAE) that logically represents adverse events associated with licensed human vaccines.

Meta-analysis of variables affecting mouse protection efficacy of whole organism Brucella vaccines and vaccine candidates.

BACKGROUND: Vaccine protection investigation includes three processes: vaccination, pathogen challenge, and vaccine protection efficacy assessment. Many variables can affect the results of vaccine protection. Brucella, a genus of facultative intracellular bacteria, is the etiologic agent of brucellosis in humans and multiple animal species. Extensive research has been conducted in developing effective live attenuated Brucella vaccines. We hypothesized that some variables play a more important role than others in determining vaccine protective efficacy. Using Brucella vaccines and vaccine candidates as study models, this hypothesis was tested by meta-analysis of Brucella vaccine studies reported in the literature. RESULTS: Nineteen variables related to vaccine-induced protection of mice against infection with virulent brucellae were selected based on modeling investigation of the vaccine protection processes. The variable "vaccine protection efficacy" was set as a dependent variable while the other eighteen were set as independent variables. Discrete or continuous values were collected from papers for each variable of each data set. In total, 401 experimental groups were manually annotated from 74 peer-reviewed publications containing mouse protection data for live attenuated Brucella vaccines or vaccine candidates. Our ANOVA analysis indicated that nine variables contributed significantly (P-value < 0.05) to Brucella vaccine protection efficacy: vaccine strain, vaccination host (mouse) strain, vaccination dose, vaccination route, challenge pathogen strain, challenge route, challenge-killing interval, colony forming units (CFUs) in mouse spleen, and CFU reduction compared to control group. The other 10 variables (e.g., mouse age, vaccination-challenge interval, and challenge dose) were not found to be statistically significant (P-value > 0.05). The protection level of RB51 was sacrificed when the values of several variables (e.g., vaccination route, vaccine viability, and challenge pathogen strain) change. It is suggestive that it is difficult to protect against aerosol challenge. Somewhat counter-intuitively, our results indicate that intraperitoneal and subcutaneous vaccinations are much more effective to protect against aerosol Brucella challenge than intranasal vaccination. CONCLUSIONS: Literature meta-analysis identified variables that significantly contribute to Brucella vaccine protection efficacy. The results obtained provide critical information for rational vaccine study design. Literature meta-analysis is generic and can be applied to analyze variables critical for vaccine protection against other infectious diseases.

Assessment and Rehabilitation of the Compromised Patient Prior to Thoracotomy.

Patients for whom pulmonary resection is anticipated often have compromised pulmonary function and decreased exercise tolerance. To avoid major morbidity and reduce mortality, identification of the high-risk patient becomes extremely important. The means of identification include rather simple testing modalities as well as those that are more complex, which report specific physiologic data. This article develops a schematic for a logical progression through the assessment of prethoracotomy patients in order that those facing a significant surgical risk might undergo pulmonary rehabilitation to improve exercise performance followed by reassessment prior to surgery.

VIOLIN: vaccine investigation and online information network.

Vaccines are among the most efficacious and cost-effective tools for reducing morbidity and mortality caused by infectious diseases. The vaccine investigation and online information network (VIOLIN) is a web-based central resource, allowing easy curation, comparison and analysis of vaccine-related research data across various human pathogens (e.g. Haemophilus influenzae, human immunodeficiency virus (HIV) and Plasmodium falciparum) of medical importance and across humans, other natural hosts and laboratory animals. Vaccine-related peer-reviewed literature data have been downloaded into the database from PubMed and are searchable through various literature search programs. Vaccine data are also annotated, edited and submitted to the database through a web-based interactive system that integrates efficient computational literature mining and accurate manual curation. Curated information includes general microbial pathogenesis and host protective immunity, vaccine preparation and characteristics, stimulated host responses after vaccination and protection efficacy after challenge. Vaccine-related pathogen and host genes are also annotated and available for searching through customized BLAST programs. All VIOLIN data are available for download in an eXtensible Markup Language (XML)-based data exchange format. VIOLIN is expected to become a centralized source of vaccine information and to provide investigators in basic and clinical sciences with curated data and bioinformatics tools for vaccine research and development. VIOLIN is publicly available at http://www.violinet.org.

Airway Management Following Tracheal Surgery.

The ability to remove longer segments of airway and to extend resections into the larynx proper has managed to create novel situations that will require attention to postoperative management. This article deals with prophylactic measures to prevent the requirement of assisted ventilation. It, however, also emphasizes various bronchoscopic and intubation techniques, which if required, will help to avoid trauma to the airway anastomosis. In addition, a variety of ventilator modalities are discussed that were developed by the author over many years at the Toronto General Hospital.

Vaxar: A Web-Based Database of Laboratory Animal Responses to Vaccinations and Its Application in the Meta-Analysis of Different Animal Responses to Tuberculosis Vaccinations.

Animal models are indispensable for vaccine research and development. However, choosing which species to use and designing a vaccine study that is optimized for that species is often challenging. Vaxar (http://www.violinet.org/vaxar/) is a web-based database and analysis system that stores manually curated data regarding vaccine-induced responses in animals. To date, Vaxar encompasses models from 35 animal species including rodents, rabbits, ferrets, primates, and birds. These 35 species have been used to study more than 1300 experimentally tested vaccines for 164 pathogens and diseases significant to humans and domestic animals. The responses to vaccines by animals in more than 1500 experimental studies are recorded in Vaxar; these data can be used for systematic meta-analysis of various animal responses to a particular vaccine. For example, several variables, including animal strain, animal age, and the dose or route of either vaccination or challenge, might affect host response outcomes. Vaxar can also be used to identify variables that affect responses to different vaccines in a specific animal model. All data stored in Vaxar are publically available for web-based queries and analyses. Overall Vaxar provides a unique systematic approach for understanding vaccine-induced host immunity.

Induction chemotherapy with mitomycin, vindesine, and cisplatin for stage IIIA (T1-3, N2) unresectable non-small-cell lung cancer: final results of the Toronto phase II trial.

PURPOSE: This is a phase II study to assess the role of induction chemotherapy in the management of stage IIIA non-small-cell lung cancer (NSCLC). We are now reporting the long-term follow-up of the Toronto phase II trial. METHODS: Sixty five patients with mediastinoscopy proven stage IIIA NSCLC received two cycles of preoperative MVP or VLB/P followed by thoracotomy followed by two further courses of chemotherapy. RESULTS: The overall response rate was 67.7% with three complete and 41 partial responders. Forty seven patients went on to thoracotomy with 35 complete resections. Pathologically 4.6% of patients had no tumour remaining. There were three postop deaths as well as five chemotherapy related deaths. Of the 35 patients completely resected 19 have recurred including eight in brain. The median survival for the entire 65 patients is 18.6 months with a 1 year survival of 66%, 5 year survival of 29% and a 10 year survival of 22%. CONCLUSIONS: The long-term survival of induction chemotherapy is maintained. The high incidence of brain recurrences warrants assessment of the role of prophylactic cranial radiation. The role of surgery for stage IIIA NSCLC following induction chemotherapy awaits further study.

Heart-lung or lung transplantation for Eisenmenger syndrome.

BACKGROUND: The optimal therapy for end-stage Eisenmenger syndrome (ES) is unknown. We analyzed the United Network for Organ Sharing/International Society for Heart and Lung Transplantation Joint Thoracic Registry to determine predictors of survival. METHODS: Univariate analysis was performed using Kaplan-Meier survival curves. Groups were compared using the log-rank test. Multivariate analysis was performed using a proportional hazards model. RESULTS: There were 605 transplants performed between 1988 and 1998. The causes of ES included atrial septal defect (ASD) in 171, ventricular septal defect (VSD) in 164, multiple congenital anomalies (MCA) in 68 and patent ductus arteriosus (PDA) in 32. Procedures included 430 heart-lung (HLT), 106 bilateral lung, and 69 single lung transplants (LT). Survival after HLT was better than after LT on univariate analysis (p = 0.002). For HLT, survival at 30 days and 1 year was 80.7% and 70.1% compared with 68% and 55.2% for LT. Diagnosis was also a significant predictor of survival (p = 0.011), being best for VSD and MCA (1-year survival 71.4% and 77.6%). There was a highly significant benefit of HLT over LT for VSD patients (p = 0.0001). Diagnosis, the combination of diagnosis and procedure, recipient age, recipient gender, donor age, ischemic time and recipient status were significant in a multivariate model. Multivariate analysis confirmed the superior prognosis of patients with VSD or MCA (p = 0.007 and p = 0.022, respectively) and suggested that the adverse effect of LT was predominately in patients with VSD (risk ratio 1.817, p = 0.035). CONCLUSIONS: This analysis suggests that ES recipients are not a homogeneous group. Patients with VSD and MCA have the best prognosis. HLT appears to offer a survival benefit for patients with ES secondary to VSD and should be re-considered as the operation of choice.

Bronchogenic carcinoma after solid organ transplantation.

BACKGROUND: This study assesses the risk of bronchogenic carcinoma after solid organ transplantation. Although the overall incidence of malignancy is increased after solid organ transplantation, the risk of bronchogenic carcinoma in the transplant population has not been systematically studied. METHODS: Among a cohort of 3,374 patients transplanted in our institution between 1985 and 2000 (1,735 kidney recipients, 930 liver, 313 heart, and 396 lung recipients), 9 patients (0.3%) had a bronchogenic carcinoma develop. Lung carcinoma occurred in 3 kidney recipients, 3 liver recipients, 2 heart recipients, and 1 lung recipient. RESULTS: Time to diagnosis after the transplant procedure ranged from 9 to 126 months (mean, 63 months). Aside from the lung transplant candidate, all recipients had a smoking history. Seven patients underwent thoracotomy and 6 had a complete resection. Tumors were classified as stage IA (n = 1), IB (n = 2), IIB (n = 2), IIIA (n = 2), IIIB (n = 1), and IV (n = 1). Genotyping demonstrated that the carcinoma arising in the lung transplant recipient originated from the donor and may have been transmitted at the time of transplantation. Two patients were alive without recurrence 21 and 42 months after the operation. CONCLUSIONS: The risk of bronchogenic carcinoma is low and occurs mainly in recipients with a smoking history. However, bronchogenic carcinoma can also be transmitted from donor lungs at the time of transplantation. Hence careful examination of chest roentgenograms, and computed tomographic chest scan if available, as well as meticulous assessment of the lung, and biopsy of any suspicious lesions, are important to limit the risk of lung cancer transmission, especially with the liberalization of donor criteria.

Monitoring and mitigating isoflurane emissions during inhalational anesthesia of mice.

Establishing a program to monitor waste anesthetic gas (WAG) in order to limit personnel exposure requires measuring the levels of WAG emitted and determining the effectiveness of scavenging methods to reduce such levels. In this study, the authors used infrared spectroscopy to measure levels of WAG emitted while anesthetizing mice with isoflurane for 15 min. They evaluated four different WAG scavenging conditions during induction and maintenance anesthesia: two conditions that used passive techniques and two that used active techniques. Isoflurane concentrations were measured at three different locations: in the operator's vicinity, at the mouse-facemask interface and in the room environment. Passive scavenging of WAG improved when chambers were purged with oxygen after induction and when a diaphragm-sealed facemask delivered a reduced anesthetic flow rate during maintenance anesthesia. Active scavenging of WAG improved when a relief intake opening was provided in the induction chamber's vacuum line, vacuum draw after induction was regulated and the anesthetic flow rate and vacuum scavenging draw were balanced during maintenance anesthesia using a facemask that separated the breathing space from the scavenging zone. Additionally, time-weighted average isoflurane WAG levels detected by personal dosimeters correlated with real-time measurements made using infrared spectroscopy. These observations contribute to the development of a substantiated program for monitoring WAG air quality.

BSim: an agent-based tool for modeling bacterial populations in systems and synthetic biology.

Large-scale collective behaviors such as synchronization and coordination spontaneously arise in many bacterial populations. With systems biology attempting to understand these phenomena, and synthetic biology opening up the possibility of engineering them for our own benefit, there is growing interest in how bacterial populations are best modeled. Here we introduce BSim, a highly flexible agent-based computational tool for analyzing the relationships between single-cell dynamics and population level features. BSim includes reference implementations of many bacterial traits to enable the quick development of new models partially built from existing ones. Unlike existing modeling tools, BSim fully considers spatial aspects of a model allowing for the description of intricate micro-scale structures, enabling the modeling of bacterial behavior in more realistic three-dimensional, complex environments. The new opportunities that BSim opens are illustrated through several diverse examples covering: spatial multicellular computing, modeling complex environments, population dynamics of the lac operon, and the synchronization of genetic oscillators. BSim is open source software that is freely available from http://bsim-bccs.sf.net and distributed under the Open Source Initiative (OSI) recognized MIT license. Developer documentation and a wide range of example simulations are also available from the website. BSim requires Java version 1.6 or higher.